The asymmetric organization of phospholipids (PL) within the bilayer of the red blood cell (RBC) membrane is a dynamic process which we hypothesize involves passive and active PL fluxes in both directions as well as tight and loose interactions of PL with membrane skeletal proteins. Variations in transbilayer mobility, PL species and membrane skeletal proteins contribute to the PL organization in RBC. We propose to investigate this hypothesis by studying PL dynamics in normal, pathologic, metabolically depleted and oxidatively damaged RBC and in vesicles obtained from these cells. Our specific aims are: 1) to determine if membrane skeletal proteins are necessary to maintain aminophospholipid asymmetry, 2) to determine if organization of choline phospholipids in the bilayer is secondary to aminophospholipid organization or if separate, identifiable mechanisms, such as passive flux, energy dependent translocation and/or membrane skeletal interactions are involved, 3) to determine what accounts for the organization of phosphoinositides in the RBC membrane and if molecular species of PI, PIP and PIP2 have different translocation rates, mobility and metabolic pathways and 4) to study pathophysiologic effects of abnormal PL organization by attempting to isolate and characterize RBC in which phosphatidylserine (PS) is on the surface of the RBC and to study the role of PL in anchoring certain RBC membrane proteins. Techniques to be used include chemically modified phospholipases, PL transfer proteins (PC specific, nonspecific and PI specific), PL transbilayer mobility probes (spin labeled and fluorescently labeled PL probes), PL fluidity probes and molecular species analysis of PL classes. New methods including FACS scan techniques will be used to try to identify in vivo RBCs which have PS on their surface so that these cells can be further characterized. Additional assays to detect surface PS will include prothrombin interactions and protein-kinase C interactions. Our studies will provide important information concerning the pathophysiologic consequences of abnormal membrane PL organization in human RBCs. In addition, since membrane PL organization is common to cells such as platelets, lymphocytes and fibroblasts, the studies described in our proposal have general application to cell biology.